Luminescence Resonance Energy Transfer (LRET) is a modification and improvement on the widely used technique of fluorescence resonance energy transfer (FRET), and can be widely used in accurately determining the distances between two sites bearing energy donor and energy acceptor respectively in a bio-molecule [1]. In LRET, one of the energy donors is a luminescent lanthanide atom enhanced by a small chelate (1):
and the acceptor is a conventional (organic) fluorophore.
LRET has great distance accuracy and range; ability to resolve multiple D-A distances; great ability to isolate signal from proteins labeled with both donor and acceptor, even in the presence of proteins labeled only with donor or only with acceptor; and less sensitivity of energy transfer to orientation of dyes (which is often unknown).
The fundamental advantages of LRET arise because the donor emission is long-lived with millisecond lifetime compared to nanosecond lifetime of acceptor or conventional dyes, is sharply-spiked (peaks of a few nanometer width), has a high quantum yield [2], and is unpolarized [3]. Also, the chelate's atomic structure has also been determined [4].
An order of magnitude greater accuracy in distance-determination is achieved with LRET because the energy transfer process is dominated by the distance between the donor and acceptor, and their relative orientations play only a minor role in determining energy transfer efficiency. (A worst case scenario is 12% uncertainty in distance determination due to orientation effect.) This advantage is because terbium donor emission is unpolarized [3]. This contrasts to FRET where the errors due to orientation effects can be unbounded. We have shown that angstrom changes due to protein conformational changes can readily be measured with LRET [5, 6].
A 100-fold improvement in signal to background (S/B) is achieved with LRET. Specifically, energy transfer can be measured with essentially no contaminating background, a stark-contrast to FRET. By temporal and spectral discrimination, donor emission and acceptor emission—both intensity and lifetime—can be independently measured. This leads to dramatically improved signal to background compared to FRET. Specifically, in LRET the acceptor emission due only to energy transfer—called sensitized emission—can be measured with no background. Contaminating background in FRET when trying to measure energy transfer via an increase in acceptor fluorescence, arises from two sources: direct excitation of the acceptor by the excitation light and donor emission at wavelengths where one looks for acceptor emission. In LRET both sources are eliminated. For example, by choosing an acceptor such as fluorescein and looking around 520 nm, donor emission is dark. By using pulsed excitation and collecting light at 520 nm only after a few tens of microseconds, all the direct acceptor emission (which has nanosecond lifetime) has decayed away. Samples that contain donor-only or acceptor-only can be spectrally and temporally discriminated against with LRET. Often when labeling proteins, particularly in living cells, one gets an unknown distribution of donor-donor, donor-acceptor, and acceptor-acceptor mixture. In FRET this makes distance-determination difficult. In LRET, sensitized emission from acceptor arises only from donor-acceptor labeled complex. Energy transfer of this D-A labeled complex can then be determined by comparing the lifetime of sensitized emission (τad), which decays with micro- to millisecond lifetime of donor that is transferring energy to the acceptor, with the donor-only lifetime (τd): E=1−τad/τd. This ability to measure energy transfer even in complex labeling mixtures is essential for the LRET studies on ion channels [5, 13].
We have published a number of papers on LRET (partially reviewed [7, 8]) showing its advantage in model systems such as DNA oligomers [9, 10], the ability to measure distance changes of an angstrom reliably even on large protein complexes such as actomyosin [11, 12], and most recently, in ion channels in living cells [5,13]. Other workers have now successfully used the technique on DNA-protein complexes [14-17], actomyosin [18], protein-protein interactions in cells [19], and detection of binding of many different biomolecules [20-22].
The current chelate-complex (1) works moderately well with both terbium and europium. The disadvantage of such chelate-complexes is that either the relatively low stability constant or fast dissociation and transmetalation kinetics limits their application in physiological environment. The lanthanide complex of 1,4,7,10-tetraazacyclododecane N,N′,N″,N′″-tetraacetic acid (DOTA) (2A) has been shown to be an excellent lanthanide chelate with a large thermal and kinetic stability constant, and has been widely used as a contrast agent in MRI imaging. Its non-reactive form of luminescent chelate, (DOTA)-cs124 (2B) has been synthesized.
But it has its limitations as well. The binding of DOTA and lanthanide ions is a kinetically slow process [23]. Furthermore, as for luminescent lanthanide probes, amine- or thiol-reactive groups facilitate attachment to a biomolecule. However, neither amine-reactive nor thio-reactive forms of DOTA-based fluorescent chelates have been reported.
The class of macrocycles known as crown ethers has been widely studied since their metal ion-coordinating capabilities were first reported by C. J. Pedersen (J. Am. Chem. Soc. 1967, 89, 7017). Derivations of the crown ether include the replacement of one or more of the ring's oxygen atoms with nitrogen atoms resulting in azacrown ethers and/or the attachment of one or more side chains to the ring to form a so-called lariat or armed crown ether. There are numerous publications on the metal-complexing properties of diazacrown ethers containing side chains attached to the nitrogen atoms of the macrocycle (see e.g. Chi et al, Bull. Korean Chem. Soc. (2002) 23(5) 688-692; Gonzalez-Lorenzo et al, Inorg Chem. (2005) 44(12): 4254-4262; Wang et al., Chinese Chemical Letters, (2003) 14(6): 579-580; Peters et al, J. Chem. Soc., Dalton Trans., (2000) 4664-4668; and I. A. Fallis, Annu. Rep. Prog. Chem. A 94 (1998) 351-387).
We have synthesized a new type of lanthanide chelate derived from diazacrown ethers. Our chelates contain two ethyliminodiacetic acid side chains and have increased ability to bind lanthanide ions.